RE: Autoradiography (Quite long reply to 2 questions)

From:

"Su, Phy-Huynh" <psu@shctampa.usf.edu>

I have recently done autoradiography using 35S-Protein mix and 35S-Sulfate
to label total protein and proteoglycan in explant culture. I've cut the
paraffin sections at 5um thick as usual. (But I've taped my microtome
anywhere I can to protect it from contamination with isotopes and it's such
a task to decontaminate it afterwards (we have strict regulations here;
instruments and benches must be cleaned up and decontaminated at the end of
the day if radioisotopes are used.)) I've used 3-aminoproyltriethoxy-silane
to sub my slides myself, and have found that to work very well. It helps to
keep my sections on glass slides. You don't want to loose sections, and in
the dark room, it's hard to know if your sections have said good-bye. I've
used Kodak NTB-2 Emulsion bought from VWR (least expensive source), and
diluted that 1:2 in water. It worked better because the gelatin coat is
thinner. I have to be very careful with the Kodak fixer, and steps after
it, because it is a powerful oxidizing agent, and it will leave trace of
brownish spots and marks on the slides. Kodak tech support Erin
(877-SIS-HELP) was of great help.
Anyway, if your researcher wants to study cell proliferating, (and I assume
either injection the animal with radioisotope, or explant culture
labelling), what about BrdU labelling??? I have injected BrdU to animals
and study cell proliferating here. It has worked beautifully. The result
is faster (developing tritium or 35S can take weeks if not months), and so
much less hassle.
That's my humble experience. Let me know if you need any help, or protocol,
or want to know how I set up my dark room, etc.... I'll be happy to help.
Su
> -----Original Message-----
> From: J. A. Kiernan [SMTP:jkiernan@julian.uwo.ca]
> Sent: Thursday, October 19, 2000 1:48 AM
> To: Cheryl Crowder
> Cc: Histonet
> Subject: Re: Autoradiography (Quite long reply to 2 questions)
>
> On Wed, 18 Oct 2000, Cheryl Crowder wrote:
>
> > ... need some help. A researcher here wants to do autoradiography on
> > mouse tissue. He wants them paraffin embedded, cut and then the slides
> > given to him for the technique. I have never done any tissue for
> > autoradiography before.
>
> > Could someone tell me how thick the sections should be cut (they are
> > tagged with small amount of tritiium)? Are they put on regular, clean
> > slides or do you use charged or coated slides?
>
> 1. Tritium emits low energy beta particles (electrons), which have
> a trajectory within tissue of about 2 um. This makes it a very
> good isotope for autoradiography because there is very little
> inaccuracy due to particles travelling obliquely into the
> emulsion and causing grains to appear over nearby non-radioactive
> places in the section.
> The short trajectory also means that if the sections are more
> than 2 um thick any tritium-labelled material in the "deeper" parts
> of the section (more than 2 um from the layer of emulsion) is not
> detected. For example, in a 10 um section you could have a 5 um
> nucleus labelled with tritiated thymidine in the bottom half of
> the section, next to the glass slide, and its beta emissions would
> never reach the emulsion. Such a nucleus would be falsely negative
> in the autoradiograph.
> For quantitative work (and all autoradiography should be
> quantitatively assessed) sections for tritium autoradiography
> should be no more than 2 um thick (1 um would be better) if all
> the sources of radioactivity are to be detected. In practice it
> isn't possible to cut large numbers of sections that are regularly
> and reliably all the same thickness, and the variability increases
> as the sections get thinner. Consequently it's more usual to use
> sections much thicker than 2 um and make allowance for the false
> negatives in the course of calculating meaningful results from
> counts of labelled cells, measurements of optical reflectance etc.
> It is still important to have all the sections the same thickness,
> and this should be whatever you are most confident with. For me this
> would be 7 or 10 um for paraffin sections. For a skilled technician
> it might well be 4 or 5 um. Below 4 um there's doubt about what
> the actual thickness is, whatever the microtome setting. (Probably
> there will be some responding flak against and for this last
> sentence!)
>
> 2. The question about the type of slide is a very good one, and I
> haven't seen any published recommendations. Adhesion is important
> not only for the sections but also for the emulsion, which can
> loosen and move during development and subsequent handling. My own
> experiences are limited to plain glass slides, but common sense
> suggests that adhesion of the emulsion (which is a suspension
> of AgBr in aqueous gelatin) should be assisted. The development
> of the autoradiograph (alkaline) is probably the most severe
> challenge to attachment of emulsion and sections to the glass.
> Slides subbed with chrome-gelatin are good for retaining
> protein-rich sections and other materials in moderately alkaline
> media. Positively charged slides (bought or made in-house with
> APES) are also good. I cannot think of a reason for not using
> subbed or APES slides for autoradiography, and I can't recall
> reading anything about it. A PubMed or Web of Science literature
> search might turn something up.
>
> 3. This email reply contains numerous unsupported statements. If
> you are interested and need references to respectable sources,
> please ask for more. I'll do my best to provide respectable
> documentation, but it may take a week or two. For exact answers
> to your questions you may need a good library mor than an
> internet listserver.
>
> Sorry to be so verbose and yet rather discouraging. The best thing
> you could do is to tell your researcher to take a week out and
> read a good autoradiography textbook. The one by the late
> A. W. Rogers is excellent.
>
> John A. Kiernan,
> Department of Anatomy & Cell Biology,
> The University of Western Ontario,
> LONDON, Canada N6A 5C1
>
>
>
>
>